The first object of the present invention is amphiphilic (hydrolipidic) complexes and more specifically proteins (and polypeptides) onto which fatty chains have been grafted. Said complexes may be qualified lipophilised proteins (and polypeptides). Other objects of the present invention are compositions, notably cosmetic, pharmaceuticals or food compositions which contain such complexes and methods for the preparation of said complexes.
The skin may be considered as an organ which separates and protects the human body from its environment. This effect of a barrier against external damaging effects is capital in order that the internal tissues suitably exert their function. External damaging effects are in fact many: luminous damaging effects (UVA, UVB, infra-red) which cause free radicals and fragmentation of the constituents of the skin, physical or mechanical damaging effects (abrasions, variations in temperature and hygrometry . . . ) which cause inflammations, chemical damaging effects (air and water pollution, contact with irritant or immunogenic elements), microbiological damaging effects (bacteria, viruses, fungi . . . ). In order to react to these various damaging effects, the skin possesses a certain number of specialised cells which sometimes form extremely well-characterised structures. This is the case of the corneocytes which, being different from keratinocytes, form a structure called the Stratum corneum which is specialised in the protection of the most internal areas of the skin. This superficial horny structure is the first protection against external damaging effects.
The use of cosmetic products and notably of hydration products also comes up against this natural barrier:
due to their small size, hydrophilic molecules of low molecular mass such as urea, lactic acid, amino acids, can penetrate via the Stratum corneum as far as in the deepest layers of the cutaneous tissues. The cosmetic effect obtained is a hydrating effect upon the deep layers of the epidermis and the dermis, an effect which is relatively short lived;
on the contrary, molecules of higher molecular mass such as proteins for example cannot cross this barrier. The Stratum corneum is in fact principally constituted of lipids (its lipid content neighbours 80% by weight), giving it a particularly hydrophobic character, totally incompatible with the hydrophilic character of most proteins used in the cosmetology field. In this case, the cosmetic effect obtained is a filmogenic effect, at times interesting for obtaining particular textures or xe2x80x9ccosmetic feelsxe2x80x9d but which remains totally and exclusively superficial.
Thus, and consequently, the hydrophilic molecules used to this day in cosmetics are cast aside by this hydrophobic structure and either stay on the surface or penetrate the dermis very deeply. From this, the horny layer and the upper layers of the epidermis are under practically no influence of the active and notably hydrating substances used up to this day in cosmetics. Now, the feeling of dryness of the skin comes from the Stratum corneum and upper layers of the epidermis. It is therefore of utmost importance to manage efficiently hydrating this structure and more generally to render said structure accessible to various hydrophilic entities.
The Applicant, within the context of the present invention, has taken on this technical problem of hydration of the skin and more generally that of the optimisation of the expression of the activity of molecules of the protein or polypeptide type upon the Stratum corneum. In order to solve said technical problem, the Applicant proposes modifying the physico-chemical character of said molecules and to thus modify the behaviour of it. The Applicant proposes in fact to generate amphiphilic complexes by grafting fatty chains onto said molecules. The trans-epidermal penetrations of such complexes are different from those of the non-complexed molecules. Their stabilisation in the upper layers of the epidermis as well as on the capillary fibre (hair) has been demonstrated. Furthermore, very interesting and unexpected cosmetic even therapeutic effects of said complexes have been observed.
It has been described in the patent application FR-A-2 671 725 about polyose-fatty acid complexes which have hydrating and emulsifying properties. These complexes are obtained by reacting, in aqueous medium, at ambient temperature, fatty acids in a reactive form with polyoses. Said polyoses can intervene in an impure form and notably in a mixture with proteins. However, in this document, no mention is made of a xe2x80x9cbinaryxe2x80x9d protein-fatty acid complex and of the interesting properties it could have . . . In any case, the polysaccharides (polyoses) having a gellifying power much greater than that of proteins, obtaining hydrating and emulsifying complexes by liophilising such proteins could not be expected. Such is all the same one of the results obtained within the context of the present invention.
It has also been described:
in the patent U.S. Pat. No. 4,234,475 a method of preparing emulsifying agents which consists in reacting, at temperatures above 200xc2x0 C., a protein and an acid, notably a fatty acid. At such temperatures, the degradation of each one of the reagents cannot be prevented, and notably the degradation of the protein (denatured and/or hydrolysed into peptides), whose properties are consequently inescapably altered;
in the application WO-A-93 22370 undecylenic acid derivatives obtained by reacting said acid in a reactive form, in an aqueous medium, at ambient temperature with a hydrophilic organic macromolecule having primary alcohol groups and/or primary amine groups, and notably with a protein. Said derivatives, very slightly fragrant, have conserved anti-fungal and anti-bacterial properties. By their approach, the expression of the activities of undecylenic acid have been above all sought-after.
Furthermore:
the application DE-A-34 22 496 describes an alcoholic disinfectant composition for the skin. Said composition contains a protein hydrolysate, a mixture of amino acids in fact,
the application EP-A-0 417 619 proposes, as a detergent showing a lesser agressivity towards the skin and the mucous membranes, the condensation products resulting from the chemical reaction between:
a hydrolysate of proteins whose average molecular mass is between 3,000 and 7,000; and
a C12-C18 fatty acid; said chemical reaction being carried out at a pH between 7 and 12 and the protein(s)/fatty acid(s) molar ratio ranging from 1/0.5 to 1/3;
the application EP-A-0 283 601 describes elastin derivatives prepared from hydrolysed elastin. Said derivatives result from a chemical coupling between said hydrolysed elastin (non-native) and a fatty acid anhydride; said fatty acid intervening, with respect to the protein (hydrolysed elastin) in a weight ratio very much lower than 1.
Said condensation products according to EP-A-0 417 619 and elastin derivatives according to EP-A-0 283 601 are not complexes within the sense of the invention. Said complexes of the invention are always elaborated in the presence of an excess of fatty acid and can be elaborated with native proteins. This is explained below.
The Applicant in fact proposes novel amphiphilic or hydrolipidic complexesxe2x80x94protein(s)/fatty chain(s) complexesxe2x80x94which, as indicated above, have very interesting and relatively unexpected cosmetic even therapeutic properties.
It is herein specified that, in the present textxe2x80x94within the context of the present inventionxe2x80x94the term protein is used to designate a xe2x80x9crealxe2x80x9d protein as well as a polypeptide (obtained eventually by synthesis).
Said complexes of the invention are, in a characteristic way, obtained from the reaction carried out at a temperature between ambient temperature and 80xc2x0 C. between:
on the one hand, one (or more) protein(s), whose average molecular mass is greater than or equal to 5,000 Daltons; and
on the other hand, one (or more) fatty chain(s), whose carbon atom number is between 4 and 30, selected from fatty acids, fatty alcohols, fatty amines and derivatives thereof, with the exclusion of undecylenic acid, the [protein(s)/fatty chain(s)] weight ratio ranging from 1/1 to 1/10 and advantageously from 1/3 to 1/5.
The reaction carried out for the coupling and/or grafting of the reagents may be chemical or enzymatic. This shall be specified further or in the present text. In any case, the reaction is carried out a temperature much lower than 200xc2x0 C., preferably lower than 100xc2x0 C. The minimisation even prevention of any degradation of the reagents and notably the intervening proteins is desired.
Said reaction is carried out with two types of reagent: on the one hand, at least one protein, on the other hand at least one fatty chain. Said fatty chains consist of fatty acids as well as fatty alcohols or fatty amines (or the derivatives of said acids, alcohols and amines).
The multiplicity and the variety of the complexes of the invention may already be insisted upon and therefore the properties that they may have; the latter depending upon the nature of the reagents (intervening protein(s) and fatty chain(s) and of their intrinsic characteristics (for example, the nature of the intervening protein, the purity of it, the molecular weight of it).
Each one of both types of reagent is specified below.
The complexes of the invention are complexes of proteins and fatty chains. The complexes obtained from amino acids are excluded from the context of said invention, whether they be purified or obtained in a mixture during the hydrolysis of a protein, as well as the complexes obtained from peptides having only 2 to 5 amino acids in their structure. The proteins which can intervene in the structure of the complexes of the invention have an average molecular mass equal to or greater than 5,000 Daltons. They consist of a chain of amino acids linked to each other by amide bonds, which has pendant amine and/or acid and/or alcohol functions.
Generally, their average molecular mass is lower than 1,000,000 Daltons. It is however in no way excluded to prepare complexes of the invention with proteins of a greater average molecular mass. Advantageously, the complexes of the invention are prepared from proteins whose average molecular mass is between 10,000 and 1,000,000 Daltons. Even more advantageously, proteins whose average molecular mass is between 20,000 and 300,000 Daltons are brought in.
In any case, the intervening proteins can be obtained by an extraction which does not destroy their structure and/or does not lower their molecular mass, or by moderate physical, chemical or enzymatic hydrolysis (said hydrolysis generating proteins whose average molecular mass is at least equal to 5,000 Daltons).
Said intervening proteins may be of animal origin (bovine, ovine, fish, shark, crustaceans, . . . ) and in this case, they may be extracted from various tissues; collagen, gelatine, albumin, ovalbumin, elastin, reticulin, fibronectin, keratin, silk, laminin, desmosin and isodesmosin, extracellular matrix proteoglycans, caseins, lactalbumin, lactoglobulins, enzymes extracted from animal tissues, etc . . . may be cited by as examples. They may be of plant origin (wheat, unicellular or multicellular algae, maize, pea, lupin, . . . )and in this case, they may be extracted from seeds, flowers, fruits, barks, gums, etc . . . ; moderated wheat, maize, cotton, lupin, pea, broad bean, almond, bean, soya, sunflower, lucerne, or oat hydrolysates or proteins, etc . . . may be cited for example.
Complexes of the invention are advantageously prepared with soya, wheat, oat or almond proteins.
As regards the second type of reagent, these are as already specified, fatty chains having from 4 to 30 carbon atoms. Advantageously, the intervening fatty chains have from 6 to 20 carbon atoms. The chains may be saturated or unsaturated, linear, branched or cyclic. They obviously have acid and/or alcohol and/or amine functions but it is in no way excluded that they have other chemical functions in their structure which intervene or do not intervene in the preparation of the complexes of the invention.
Said fatty chains may notably be selected from the heptanoic, octanoic, decanoic, lauric, myristic, palmitic, stearic, ricinoleic, oleic, linoleic, linolenic fatty acids; the corresponding fatty alcohols and fatty amines; the derivatives of said fatty acids, fatty alcohols and fatty amines; and mixtures thereof.
Advantageously, complexes of the invention are prepared:
with lauric, stearic or palmitic acids and notably a mixture of stearic and palmitic acids;
with laurylamine or hexadecylamine;
with decylalcohol.
It has been seen that said fatty chains consist of fatty acids, fatty alcohols or fatty amines (or their derivatives). For the preparation of the complexes of the invention chemically, said fatty acids optionally intervene in reactive forms (more reactive), and notably as halides (chlorides, bromides, iodides, . . . ), anhydrides or derivatives of anhydrides.
Within the structure of the complexes of the invention, the [protein(s)]/[fatty chain(s)] weight ratio ranges from 1/1 to 1/10 and preferentially from 1/3 to 1/5.
The protein(s) are in fact always allowed to react with a more or less large excess of fatty chain(s) with the aim of creating covalent bonds but also of the ionic, hydrogen and Van der Waals type.
Generally and furthermore, at the end of the reaction, the unreacted fatty chains which are not bound to the protein are not recovered, the isolation of xe2x80x9cbinaryxe2x80x9d complexes of the pure protein(s)-fatty chains type is not attempted. Thus, the complexes of the invention generally consist of xe2x80x9cbinaryxe2x80x9d complexes of the type indicated above in a mixture with non-bound fatty chains; in other words, they consist of the product of the coupling reaction in a mixture with unreacted (un-coupled) fatty chains.
Said complexes constitute the first object of the present invention. The compositions, notably cosmetic, pharmaceutical or food compositions, containing them constitute the second object of the said invention.
Said compositions generally contain from 0.01 to 40% by weight of such complex(es) and advantageously from 0.1 to 10% by weight.
More specifically, the notably cosmetic, pharmaceutical or food compositions which contain at least one protein as active ingredient make up an integral part of the present invention; said protein which intervenes, being at least in part (even in totality) as a complex such as described above. For the elaboration of said compositions, said complex may be used purified (isolated from the reaction mixture in which it was synthesised) or as a mixture with one and/or the other of the reagents which intervened in its synthesis. According to this second variant, the reaction mixture (at the end of the reaction) is advantageously used which contains said complex and the unreacted reagents (principally fatty chains insofar as they intervene in excess).
The compositions wherein said complexes intervene as emulsifying agents also make a part of the invention.
In any case, it was noted in a surprising way that the complexes of the invention have hydrating and emulsifying properties. This is relatively unexpected insofar as the person skilled in the art cannot ignore that the proteins have a capacity to trap water which is much lower than that of polysaccharides and insofar as said capacity, which is relatively low in the absolute, should have been affected by the lipophilisation of said proteins.
In addition to these hydrating and emulsifying properties, which are relatively unexpected, the complexes of the invention have revealed to have other properties which are totally unexpected.
It is as such that a soluble wheat protein having an average molecular mass of 100,000 Daltons, onto which stearic and palmitic acid chains have been grafted, have extremely strong skin restructuration properties, which enable one to envisage the use of this lipophilised protein (complex in the sense of the invention) in applications wherein a destructuration of the epidermis is observed (physico-chemical damaging effects or skin ageing . . . ).
Similarly, a soluble almond protein having an average molecular mass of 30,000 Daltons, onto which stearic and palmitic acid chains have been grafted, has the property of calming moderate to strong sunburn, which enables envisaging the use of this lipophilised protein (complex in the sense of the invention) in sun or after-sun formulations.
Similarly, an insoluble laminary protein having an average molecular mass of 10,000 Daltons onto which caprylic acid chains have been grafted has the property of inhibiting a certain number of micro-organisms, which enables one to envisage the use of this lipophilised protein (complex in the sense of the invention) in applications wherein the destruction of micro-organisms is envisaged (anti-acne effects, anti-dandruff effects, anti-body odour effects, natural preservative . . . ).
It has previously been insisted upon the diversity of the complexes of the invention. The interest of such a diversity is herein referred to.
Furthermore, it is recalled herein that the properties of the complexes of the invention, whether they are more or less unexpected, are expressed as expected, on the Stratum corneum, by their lipophilisation.
The compositions of the invention therefore consist essentially of cosmetic compositions. It is not excluded that these are therapeutic, food or dietary compositions which are particularly efficient on the mucous membranes.
According to its third aspect, the invention relates to a method of preparing amphiphilic (hydrolipidic) complexes described above. Said method characteristically comprises the reaction, at a temperature between ambient temperature and 80xc2x0 C., if need be in aqueous medium or solvent medium, between at least one protein of the aforementioned type and at least one fatty chain of the aforementioned type, said reagents intervening in a [protein(s)/fatty chain(s)] weight ratio between 1/1 and 1/10, advantageously between 1/3 and 1/5. Said reaction may be qualified a grafting reaction or more exactly a coupling reaction (insofar as it does not generate only covalent bonds between the reagents).
Said reaction is carried out at a relatively low temperature. The degradation of the reactive proteins is thus minimised. The reaction brings in or does not bring in a solvent, aqueous medium or organic solvent. The intervention of such a solvent may be done away with if, at the temperature of the reaction, the reagents are liquid.
At the end of the reaction, the xe2x80x9cbinaryxe2x80x9d complexes are generally not isolated. They are found thus in a mixture principally with the unreacted fatty chains.
It is generally desired to adjust the pH of the complexes obtained in order to render it compatible with the later, notably cosmetic applications. The pH is adjusted to values between 2 and 10 and more particularly between 5 and 7. To this end, neutralising agents are used which are selected from:
inorganic bases (such as KOH, NaOH, Ca(OH)2. . . );
metal bases (as hydroxide, carbonate . . . );
organic bases (citrate, phosphate, borate, acetate, TRIS . . . buffers; C1-C6 amines or alkylamines: triethanolamine, aminomethylpropane . . . ).
After preparing the complexes whose pH, if need was, adjusted to pHs compatible with their later use (said pH is advantageously adjusted by dispersion of said complexes in the aqueous phase), it is possible to dry them by atomisation, lyophilisation, dehydration under vacuum . . . Said dried complexes, or directly obtained without water, may be then made into a form, notably in the form of turnings.
The coupling reaction carried out may be carried out chemically or enzymatically. The enzymatic route is, within the context of the present invention, totally original.
According to said chemical route, it is possible to:
+react the fatty chainsxe2x80x94fatty acids, fatty alcohols, fatty aminesxe2x80x94under conventional conditions of peptide synthesis; i.e. in the presence of bifunctional agents, such as diimides;
+react the fatty acids in reactive (more reactive) forms i.e. react halides (chlorides, bromides, iodides . . . ) of fatty acids, fatty acid anhydrides, fatty acid anhydride derivatives . . .
According to said original enzymatic route, the proteins are coupled to the fatty chains in the presence of an enzyme, generally at a temperature between 30 and 70xc2x0 C. Advantageously said temperature is between 50 and 60xc2x0 C. Advantageously, the intervening enzyme is an acyltransferase. According to three variants of this enzymatic route, said enzyme is a lipase, notably selected from Mucor miehei lipase, pig pancreas lipase, Rhizopus arrhizus lipase, Candida lipase, Bacillus lipase and Aspergillus lipase, or a protease, notably consisting of papaine, or an amidase. Such an enzymatic reaction ensures, as the chemical reactions recalled above, the grafting of fatty chains onto the proteins. Said fatty chains, when they are fatty acids, can intervene as esters (including esters of glycerides). The enzyme present in the reaction medium ensures, firstly, the transesterification.
The reaction carried out, chemical or enzymatic, ensures the coupling by generating ester and/or amide covalent bonds. It has been seen that said coupling also brings in ionic bonds, hydrogen bonds, Van der Waals type forces . . .
The reaction is advantageously carried out with a water activity of the reaction medium (aw) between 0.2 and 1 and advantageously between 0.3 and 0.7.
The methods of preparing compositions of the invention, notably cosmetic, pharmaceutical and food compositions containing hydrolipidic complexes also make up a part of the invention. They principally consist in mixing the active ingredient with an appropriate excipient. It has been seen that said active principle could have emulsifying properties. This can reveal to be particularly interesting. The intervention of any synthetic emulsifying agent may thus be limited, even eliminated.